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Proline is a unique amino acid. Chemists will tell you it’s actually an α-imino acid, but nomenclature aside, proline is the only one of the standard 20 that is cyclic and exists in cis and trans isoforms. Switching between those two configurations can profoundly change a protein’s secondary structure, and enzymes that have evolved to flip this proline switch, the peptidyl-prolyl isomerases (PPIs), exert control over a plethora of cellular processes. One isomerase, Pin1, has been linked to Alzheimer and other neurodegenerative diseases via amyloid-β and tau (see ARF related news story). Now, two more PPIs join the fold.

FK binding proteins (FKBPs) are PPIs that bind the immunosuppressant drug FK506; they can regulate tau and α-synuclein and may exacerbate their toxicity, according to a recent publications in PNAS and the Journal of Neuroscience. “These papers provide further evidence that prolyl isomerases play an important role in age-dependent neurodegeneration, such as in Alzheimer and Parkinson diseases. They also suggest FKBPs as novel drug targets for some neurodegenerative disorders,” Kun Ping Lu, Beth Israel Deaconess Medical Center, Boston, told ARF. Lu was not involved in either study. He also thinks FKBPs may turn out to have more profound effects on neurons than has been appreciated. A recent paper in Neuron linked the same two isomerases, FKBP12 and FKBP52, to activation of calcium channels that control neuronal growth cones.

Also called immunophilins, FKBPs are best known for their role in immunology, though they have long been suspected of influencing neurons as well. Of the 15 members of the isomerase family, four, including FKBP12 and FKBP52, are highly enriched in the brain, while compounds that bind them, such as FK506, have been shown to be neuroprotective. That protection was initially thought to be related to immunosuppressant activity, so in the late 1990s, it came as a surprise when scientists found FKBP ligands that protect neurons but have no effect on the immune system. The immunosuppression and anti-isomerase activity turned out to be distinct, raising the possibility that only the latter was the basis for neuroprotection. However, the peptidyl-prolyl isomerase(s) in question remained elusive, noted Lu. Now, these three papers indicate that the enzyme activity of FKBPs plays a role in neuron function.

Toying With Tau
In the PNAS paper, published February 9, Etienne-Emile Baulieu and colleagues at INSERM, Paris, France, report that FKBP52 binds and regulates the microtubule binding protein tau. The work was done in collaboration with Michel Goedert at the Medical Research Council, Cambridge, UK. First author Béatrice Chambraud and colleagues had previously found that FKBP52 binds to tubulin, the building block of microtubules, and prevents its polymerization (see Chambraud et al., 2007), and they were curious to know if the isomerase also binds microtubule-associated proteins (MAPs) such as tau. Chambraud used an affinity chromatography strategy, with FKBP52 as bait, to capture proteins in cytosolic microtubule preparations from rat brain. The approach reeled in tau, but not Map1b or 2, suggesting the FKBP52-tau interaction might be relatively specific. FKBP52 and tau also co-immunoprecipitated from microtubule preps. The isomerase bound recombinant human tau in vitro, with somewhat higher affinity for the hyperphosphorylated form, which is most likely to form the neurofibrillary tangles found in a wide range of neurodegenerative tauopathies. In an in vitro microtubule experiment, FKBP52 prevented tau-induced formation of tubulin polymers. This effect was seen with any of the six isoforms of human tau driving polymerization.

Turning to cells, the authors found FKBP52 colocalized with tau in primary cortical neurons from rats and in PC12 cells. The colocalization was strongest at distal portions of axons and at growth cones, where FKBP52 strongly accumulated. When the researchers overexpressed the isomerase in PC12 cells, it prevented accumulation of tau in response to nerve growth factor (NGF), and it prevented NGF-induced increase in neurite outgrowth.

Where does the proline twisting come into all this? Chambraud and colleagues do not have evidence that FKBP52 actually isomerizes prolines on tau, though they are currently in the midst of experiments to test this hypothesis. “But from indirect results, we really believe that there is a change of conformation of tau under the influence of FKBP52,” Baulieu told ARF.

Irrespective of isomerization, the researchers conclude that FKBP52 acts as an “anti-tau,” and suggest that modulating its activity with non-immunosuppressive analogs of FK506 may be one way of reducing the pathogenic effects of misfolded tau. “We are opening a new chapter in the study of the regulatory aspects of tau biology, because that is dependent on the amount and the conformation of FKBP52, which is itself dependent on ligands of FKBP52,” Baulieu told ARF. Lu agrees that blocking FKBP52 might offer an opportunity for tackling tauopathies. He noted that, unlike its cousin Pin1, which acts on tau to promote microtubule assembly and prevent tau toxicity, FKBP52 may promote tauopathies because it destabilizes microtubules. That “is especially exciting given that FKBP inhibitors, which are clinically used as immunosuppressive drugs, have been shown to have neuroprotective effects,” he suggested in a written comment (see full comment below).

Scuppering Synuclein
PPI inhibitors might also be worth exploring for synucleinopathies. Writing in the February 17 Journal of Neuroscience, researchers led by Veerle Baekelandt at the Katholieke Universiteit Leuven, Belgium, report that FKBP52 and the closely related FKBP12 promote aggregation of α-synuclein and cell death, and that FK506 prevents this. The compound also prevents α-synuclein aggregation in mouse brain.

First author Melanie Gerard and colleagues used a neuronal cell culture model to test the role of FKBPs in α-synuclein aggregation. In this model, the scientists expose SHSY5Y neuroblastoma cells to hydrogen peroxide and ferric chloride to induce oxidative stress. After three days of continuous treatment, cytoplasmic aggregates of α-synuclein appear that can be detected with antibodies or thioflavin S staining.

Gerard and colleagues found that FK506 dose-dependently reduced the number of α-synuclein inclusions in this stress model, suggesting that FK506 binding proteins contribute to α-synuclein toxicity. To help identify those involved, the authors chose to knock down FKBP12 and FKBP52, two of the four FKBPs that are abundant in the brain. (The other two, FKBP38 and FKBP65, occur in mitochondria and endoplasmic reticulum, respectively, hence are probably less likely to interact with the cytoplasmic synuclein, the scientists reasoned.) Transiently knocking down either FKBP12 or 52 alone reduced oxidative stress-induced α-synuclein aggregation by 40 to 60 percent (the authors did not report on knocking down both proteins together). Stably reducing FKBP12 achieved even better results. By introducing a lentiviral vector expressing a short-hairpin RNA that silenced FKBP12 mRNA, the researchers almost completely blocked the accumulation of α-synuclein and accompanying nuclear condensation, which is a hallmark of programmed cell death. In the reverse experiment, they found that overexpressing either of the FKBPs exacerbated synuclein aggregation and death, and that FK506 could rescue the effect.

These assays indicate a role for FKBP12 and 52 in promoting α-synuclein aggregation in stressed cell cultures, but what about in the brain? Two pieces of evidence suggest some physiological relevance. First, the researchers found that FKBP12 colocalized with inclusions in aged α-synuclein transgenic mice. Second, they found that FK506 improves outcomes when given orally to mice that received an injection into the striatum of a lentiviral vector expressing the α-synuclein gene. After five months, those animals test positive for α-synuclein- and ubiquitin-containing inclusions characteristic of the Lewy bodies found in Parkinson disease, but animals receiving FK506 had slightly fewer α-synuclein-positive inclusions and also more surviving cells.

Together, the two papers suggest roles for FKBPs in both tau- and synucleinopathies. “These two papers finally connect FKBPs to disease,” suggested Lu. “Before, they were considered in the context of general neuroprotection or for stroke, but now they are presented in the context of age-dependent neurodegeneration.” In keeping with this, Baekelandt and colleagues propose FKBPs as novel targets for Parkinson’s drugs and note that because FKBP ligands are clinically approved as immunosuppressants, drug development may be straightforward. Such compounds might be spared nasty side effects as well, since FKBP12 and 52 are unusually abundant in neurons and not found in other cells.

Gerard and colleagues also do not present evidence that FKBPs actually isomerize α-synuclein (α-SYN) prolines, but they note that the protein contains five of the amino acids, and they are all in the C-terminal end of the protein, which seems to be the driving force for aggregation. “If PPI-ases induce certain folds in α-SYN, the deregulation of their activity in conditions of oxidative stress might erroneously favor those conformations of α-SYN that lead to aggregation and toxicity,” write the authors.

A solid link between proline isomerization and neurobiology appeared in last November’s Neuron. Researchers led by Paul Worley and Guo-Li Ming at Johns Hopkins University, Baltimore, Maryland, reported that during development, FKBP52 helps steer neuronal growth cones in response to chemical cues. First author Sangwoo Shim and colleagues found that, in Xenopus, FKBP52 regulates the opening of the TRPC1 cation channel, which mediates calcium influx in response to netrin-1, a chemoattractant for neuronal growth cones. The authors used nuclear magnetic resonance spectroscopy to prove that FKBP52 isomerizes prolines in the N- and C-terminals of the cation channel. They found that FKBP12 can isomerize the same prolines (P20 and P645) and also regulate TRPC1, but in spontaneous fashion and not in response to growth cues.

The action of FKBPs on TRPC1 may also be relevant to human disease. In contrast to Xenopus, the authors found that FKBP52 regulates responses to repulsive cues in mammalian hippocampal neurons. Specifically, they found that FKBP52 is essential for myelin-associated glycoprotein (MAG)-induced growth cone repulsion. MAG and related proteins can block axon regeneration and are being studied in the context of spinal cord injuries in mammals. “FKBP52 may provide a rational target for new medicinal chemistry directed toward promotion of regeneration and neuron protection in the adult central nervous system,” suggest the authors.—Tom Fagan

Comments

Comments on News and Primary Papers

Distinct Contribution of Peptidyl Prolyl Cis-trans Isomerases to Tau Functions
Some peptidyl prolyl bonds in certain proteins such as tau can exist in two completely distinct cis and trans conformations, whose conversion can be greatly accelerated by peptidyl prolyl cis-trans isomerase (1,2). A increasing body of evidence indicates that the peptidyl prolyl cis-trans isomerization can act as a novel molecular timer to regulate the amplitude and duration of cellular processes (1,2). This new paper by Chambraud et al. reveals an interesting novel role for the peptidyl prolyl cis-trans isomerase FKBP52 in regulating the function of tau, a microtubule-binding protein that plays a major role in the development of Alzheimer disease and related tauopathies.

FKBP52 is a member of the FKBP (FK506-binding protein) family of peptidyl prolyl cis-trans isomerases and has recently been shown to control chemotropic guidance of neuronal growth cones via regulation of TRPC1 channel opening in the developing spinal cord. Chambraud et al. reported that FKBP52 interacts with tau, especially when the latter is in its hyperphosphorylated form, and that FKBP52 and tau colocalize in the distal part of the axons of cortical neurons.

Tau functions at two major stages in the brain: during brain development and brain aging. Dynamic tau phosphorylation occurs during embryonic development, which may play an important role in refining or maintaining neuronal structure and function, whereas tau hyperphosphorylation affects tau function and degradation, leading to tangle formation and neurodegeneration in tauopathies (4). Interestingly, Chambraud et al. found that FKBP52 inhibits the ability of recombinant tau to promote microtubule assembly in vitro. Moreover, overexpression of FKBP52 in differentiating PC12 cells prevents tau accumulation and reduces neurite outgrowth. These results demonstrate that FKBP52 has an inhibitory effect on neuronal differentiation/development and suggest that FKBP52 may be a promoting factor of tauopathy. The latter possibility is especially exciting given that FKBP inhibitors, which are clinically used as immunosuppressive drugs, have been shown to have neuroprotective effects.

Strikingly, these effects of FKBP52 on tau are in sharp contrast to those of Pin1, an another, but distinct peptidyl prolyl cis-trans isomerase (5-8). Pin1 binds to and isomerizes the phosphorylated Thr231-Pro motif in tau to restore tau ability to bind microtubules and to promote their assembly (9), to facilitate tau dephosphorylation by PP2A (10,11), and to promote tau degradation (12). The impact of Pin1 on tauopathy and neurodegeneration has been well established. Pin1 is inhibited by multiple mechanisms in the Alzheimer brain in humans, and deletion of Pin1 in mice causes progressive age-dependent tauopathy (2,10). Moreover, postnatal neuronal Pin1 overexpression effectively inhibits the tauopathy phenotype induced by overexpression of wild-type tau in transgenic mice (1,2). These results demonstrate a pivotal role of Pin1 in protecting against tauopathy in Alzheimer disease.

Although both Pin1 and FKBP52 have peptidyl-prolyl isomerase (PPIase) activity and a specific protein-protein interaction domain (2), they seem to have the opposite effects on tau functions. It is not clear why they have such opposite effects on tau. Notably, Pin1 specifically isomerizes only phosphorylated Ser/Thr-Pro motifs and acts only on phosphorylated tau, but FKBP52 has much less activity towards a Ser/Thr-Pro motif after phosphorylation (7), and Chambraud et al. show it can inhibit the function of non-phosphorylated tau. One interesting possibility is that FKBP52 might act directly on non-phosphorylated tau, although its binding to tau seems to be enhanced by tau hyperphosphorylation. Future studies on how FKBP52 regulates tau protein conformation and function, and whether manipulating FKBP52 affects tauopathy in animal models might help offer new insight into tau regulation and provide potential new approaches to inhibit tauopathies.

The role of FKBPs in neuronal development and protection has been of interest since the discovery that FKBP inhibitors, such as FK506, possess neuroprotective and neuroregenerative qualities, similar to those of cyclosporine A (Sharkey and Butcher, 1994; Giordani et al., 2003; Sosa et al., 2005). FK506 has also been shown to augment the effects of nerve growth factor (NGF) in promoting neurite outgrowth in PC12 cells and rat dorsal root ganglion explants (Lyons et al., 1994). FK506 has also been shown to promote the regeneration of damaged sciatic nerves in vivo (Gold et al., 1995). It was thought that the regenerative powers of FK506 are dependent on the interaction of FKBP52 with the steroid receptor complex in neuronal cells. Disruption of this interaction by addition of FK506 releases the constituent components p23 and HSP90, which activate downstream signaling pathways and the neuroregenerative response (Gold et al., 1999). However, other evidence suggests that FKBP38 could be responsible for both the neuroprotective and neurotrophic properties of FKBP inhibitors (Edlich et al., 2006).

This new study demonstrates the interaction of FKBP52 with tau protein (Chambraud et al., 2010) and may shed new light on the role of FKBP52 in neuroprotection. It remains to be seen which region of FKBP52 is required for binding HP-tau. I find it unlikely that the peptidyl-proline isomerase (PPIase) domain of FKBP52 interacts specifically with hyperphosphorylated tau, since the proline-binding regions of the FKBP PPIase domains lie in a hydrophobic pocket and show a preference for bulky hydrophobic residues (such as leucine or phenylalanine) preceding the proline in peptide substrates (Van Duyne et al., 1993). Pin1, however, uniquely contains a cluster of basic residues within the active site of its PPIase domain and an N-terminal WW-domain, which convey substrate specificity for a phosphorylated serine or threonine residue preceding the proline. The interaction of Pin1 with tau is dependent on phosphorylation of Thr231, an interaction that may be a critical factor in the prevention of Alzheimer disease (Lu et al., 1999, Thorpe et al., 2004).

The contrasting effects of FKBP52 and Pin1 on tau function are illustrated in microtubule polymerization assays. Lu et al. (1999) demonstrated that Pin1 promotes microtubule assembly by promoting the dephosphorylation of p-tau. Chambraud et al., on the other hand, show that addition of FKBP52 to these assays inhibits microtubule assembly. This suggests that the two PPIases exert functionally distinct, rather than degenerate, effects on tau function. The contrasting effects of each protein on the properties of tau suggest that a fine balance of FKBP52 and Pin1 function is required to prevent the onset of disorders such as Alzheimer disease.